Abstract HIVpeptidesprocessedanddisplayedbyHIV-infectedcellsconstitutesthetriggerforTcellimmunerecognition. ThepaucityofconventionalHIVpeptidesdisplayedbyHIV-infectedcells(5%oftotalMHC-peptides)contrasts with the large number of documented HIV immune responses. As these responses are identified with MHC- peptide-tetramers,theybypassallstepsofantigenprocessingandkineticsofpeptidepresentationdefiningthe surfacepeptidomeofinfectedcells.Toidentifytargetsofimmuneclearanceoftrulyinfectedcells,itiscriticalto understand HIV antigen processing, and specifically steps occurring early in the cytosol of CD4 T cells where virus is delivered. We hypothesize that an unconventional mechanism of antigen processing yielding spliced peptidesinthecytosolgeneratesnovelmarkersofearlyeventsofinfectionofCD4Tcells. HIVpeptidesdisplayedby MHC come from the intracellular degradationof HIV antigensbyproteasomesand other peptidases, generating the pool of peptides available for MHC presentation. Recent studies in cancer, Lysteria and self proteomes identified splicedpeptides,twonon-contiguous degradation peptides reassemble insidetheproteasomeduringantigenprocessing.Cancerantigen-derivedandLysteria-derivedsplicedepitopes elicited cytolytic CD8 T cell responses. One cancer spliced peptide in gp100 was presented by MHC as abundantlyasaconventionalgp100epitopeandtheimmunogenicityofoneLysteriaproteinsolelyreliedontwo spliced peptides. Thus, spliced peptides are a significant partof the repertoire of MHC-peptides (4-25%ofthe selfpeptidome),andCD8Tcellsspecificforsplicedpeptidesclearabnormalcells. We present the first identification of HIV-derived spliced peptide during the cytosolic degradation of a HIV fragment in CD4 T cell extracts, which are also produced endogenously in primary HIV-infected CD4 T cells. Sincepeptidesplicingrequiresproteasomes,itwouldoccurduringthedegradationofincomingvirions,orduring thedegradationofHIVdefectivetranslationproducts,theearliestnewlysynthesizedHIVtranslationproducts.If peptidesplicingrepresents4-25%ofproteasomaldegradationproducts,onewouldatleastdoubletheknown pool of HIV-derived MHC-peptides displayed by infected cells, which would guide the identification of novel protectiveimmuneresponsesandopennewopportunitiesforimmunogendesign. Through a combination of biochemical and massspectrometry assays to follow antigen degradation,to isolate intracellular and MHC-peptides combined with powerful bioinformatics tools, we propose to 1) identify unconventional HIV-derived splicedpeptidesprocessed anddisplayed by HIV-infected CD4 T cells, 2) assess the immunogenic potential of HIV-derived spliced peptides. This proposal builds on the expertise of the PI in antigen processing and mass spectrometry in collaboration with bioinformatics experts, and the access to samplesfromcohortsofHIV-infectedpersonsfromtheRagonInstitute.